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The Journal of Immunology, 2004, 173: 6089-6097.
Copyright © 2004 by The American Association of Immunologists

Antigen Targeting to CD11b Allows Efficient Presentation of CD4+ and CD8+ T Cell Epitopes and In Vivo Th1-Polarized T Cell Priming1

Géraldine Schlecht*, Jirina Loucka{dagger}, Hossain Najar*, Peter Sebo{dagger} and Claude Leclerc2,*

* Unité de Biologie des Régulations Immunitaires, Institut National de la Santé et de la Recherche Médicale E 352, Institut Pasteur, Paris, France; and {dagger} Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Prague, Czech Republic


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bordetella pertussis adenylate cyclase (CyaA) is an invasive bacterial toxin that delivers its N-terminal catalytic domain into the cytosol of eukaryotic cells bearing the {alpha}M{beta}2 integrin (CD11b/CD18), such as myeloid dendritic cells. This allows use of engineered CyaA for targeted delivery of CD8+ T cell epitopes into the MHC class I pathway of APC and induction of robust and protective cytotoxic responses. In this study, we demonstrate that CyaA can efficiently codeliver both a CD8+ T cell epitope (OVA257–264) and a CD4+ T cell epitope (MalE100–114) into, respectively, the conventional cytosolic or endocytic routes of processing of murine bone marrow-derived dendritic cells. Upon CyaA delivery, a strong potentiation of the MalE100–114 CD4+ T cell epitope presentation is observed as compared with the MalE protein, which depends on CyaA interaction with its CD11b receptor and its subsequent clathrin-mediated endocytosis. In vivo, CyaA induces strong and specific Th1 CD4+ and CD8+ T cell responses against, respectively, the MalE100–114 and OVA257–264 epitopes. These results underscore the potency of CyaA for design of new vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Exogenous and endogenous Ag are processed in APC by two distinct conventional pathways to generate peptides for MHC-restricted presentation (1). Exogenous Ag are taken up and processed into peptides by proteases along the endocytic pathway, while endogenous proteins are degraded by the proteasome within the APC cytoplasm. The peptides generated by this processing bind respectively to nascent MHC class II (MHC II) or MHC class I (MHC I) molecules (2, 3). Exogenous cell-associated or particulate Ag can also be cross-presented by MHC I molecules through alternative pathways of processing (4, 5, 6, 7). One approach to induce CTL responses against exogenous Ag takes advantage of the capacity of certain proteins, mainly bacterial toxins, to enter the cytosol of APC, where they are processed by the proteasome and are then presented by MHC I molecules. Thus, several vaccinal strategies using recombinant bacterial toxins have been designed to generate CTL responses against exogenous Ag (8, 9, 10, 11).

Bordetella pertussis adenylate cyclase (CyaA)3 delivers directly its N-terminal catalytic (adenyl cyclase (AC)) domain into the cytosol of eukaryotic cells bearing the {alpha}M{beta}2 integrin (CD11b/CD18) (12, 13). This receptor is present on macrophages, neutrophils, and NK cells, but also on certain dendritic cell (DC) subsets (14). CD8+ T cell epitopes inserted into the AC domain of a genetically detoxified CyaA were, indeed, shown to be delivered into the cytoplasm of CD11b+ DC both in vitro and in vivo (15). This targeted delivery of CD8+ T cell epitopes into the MHC I pathway allows the induction of robust and protective CTL responses (16, 17, 18, 19) that are strongly polarized toward the Th1 profile (20). This CD8+ T cell activation does not require CD4+ T cell help, nor the CD40 signaling (15). Thus, CyaA appears to be a safe and potent vehicle for in vivo Ag delivery to CD11bhigh DC (21), leading to CD8+ T cell priming. However, the generation of optimal CD8+ T cell responses for prophylactic and therapeutic purposes may require the simultaneous activation of CD4+ T cell responses (22, 23, 24, 25, 26, 27). Therefore, successful vaccinal strategies will need the simultaneous delivery of both CD4+ and CD8+ T cell epitopes to APC for efficient T cell priming.

In this study, we examine the capacity of the AC domain of CyaA to simultaneously deliver CD4+ and CD8+ T cell epitopes into the MHC I- and II-restricted presentation pathways, respectively. Recombinant detoxified CyaAs carrying in their AC domains the MalE100–114 CD4+ T cell epitope (CyaA-MalE), the OVA257–264 CD8+ T cell epitope (CyaA-OVA), or both epitopes (CyaA-MalE-OVA) were constructed, and their capacity to deliver epitopes for MHC-peptide complex formation was monitored in vitro and in vivo.

Our results demonstrate that the AC domain of the CyaA is in vitro delivered into both MHC I and II presentation pathways. A strong potentiation of CyaA-MalE MHC II-restricted presentation is observed as compared with the presentation of MalE protein or peptide, which is dependent on CD11b-CyaA interaction. Using various inhibitors as well as TAP-deficient DC, we demonstrate that after its interaction with CD11b, the CyaA AC domain can either be translocated into DC cytosol to be processed along the conventional MHC I processing pathway or be endocytosed and degraded for MHC II-restricted presentation along the endocytic route of processing. In vivo, CyaA-MalE-OVA simultaneously induced CTL responses against the OVA257–264 peptide, as well as Th1 cytokine production specific for both MalE100–114 and OVA257–264 epitopes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Six- to 10-wk-old female C57BL/6 (H-2b) mice from Iffa Credo (L’Arbresle, France) were used. TAP1 knockout mice (TAP–/–) (28) onto a C57BL/6 background were a gift from A. Bandeira (Institut Pasteur, Paris, France) and were bred in our animal facilities. MHC II–/– mice on the H-2b background (B6.129-H2dlAb1-Ea/J) were obtained from The Jackson Laboratory (Bar Harbor, ME). Wild-type F1 mice (C57BL/6 x 129sv) were used as control mice.

Peptides and proteins

The synthetic peptides SIINFEKL and NGKLIAYPIAVEALS, corresponding respectively to the CD8+ T cell epitope encompassing the OVA residues 257–264 (29) and to the CD4+ T cell epitope corresponding to Escherichia coli MalE protein residues 100–114 (30), were purchased from Neosystem (Strasbourg, France). MalE protein was a kind gift of J. M. Clément (Institut Pasteur), and OVA was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

Construction, production, and purification of rCyaA toxoids with inserted CD4+ MalE100–114 and CD8+ OVA257–264 epitopes

To construct the hybrid cyaA alleles coding for the CyaA proteins carrying simultaneously the MalE100–114 and the OVA257–264 epitopes, appropriate unique restriction sites were used for recombination of cyaA alleles carrying oligonucleotide inserts coding for either the MalE100–114 epitope (31) or the OVA257–264 epitope (32). The insertion and the orientation of oligonucleotides in cyaA gene were verified by restriction analysis of plasmids. All constructs were genetically detoxified by insertion of a dipeptide sequence at the 188 position.

The E. coli strain XL-1 Blue (Stratagene, La Jolla, CA) was transformed with the constructed plasmids containing the accessory gene cyaC required for posttranslational acylation of CyaA (32). The cells were grown, as described previously (32), and the expression of recombinant proteins was induced by adding 1 mM isopropyl {beta}-D-thiogalactoside. The CyaA proteins were extracted with 8 M urea (33) and purified by DEAE-Sepharose and phenyl-Sepharose chromatographies (34). The length and homogeneity of purified toxins were verified by 7.5% SDS-PAGE, and their concentrations were determined by Bradford method. The rCyaA used in this study bear the NGKLIAYPIAVEALS insert between aa 108 and 109 (CyaA-MalE), the SIINFEKL peptide between aa 336 and 337 (CyaA-OVA), or both sequences in their respective insertion sites (CyaA-MalE-OVA).

Purified CyaA E5, a genetically detoxified CyaA without insert, was kindly provided by D. Ladant (Institut Pasteur) and was used as a negative control.

Culture medium

Complete medium (CM) consisted of RPMI 1640 containing L-alanyl-L-glutamine dipeptide supplemented with 10% FCS (Valbiotech, Paris, France), 5 x 10–5 M 2-ME, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin).

Cell lines

The H-2b-restricted hybridoma CRMC3, specific for the NGKLIAYPIAVEALS sequence of the MalE protein from E. coli, was generated, as previously described (30), and was maintained in CM. B3Z (35), the CD8+ T cell hybridoma specific for the Kb-restricted SIINFEKL peptide, was a generous gift of N. Shastri (University of California, Berkeley, CA), and was maintained by adding 1 mg/ml G418 and 400 µg/ml hygromycin B to the CM. The EL-4 thymoma was obtained from American Type Culture Collection (Manassas, VA).

Bone marrow-derived DC (BMDC) generation

BMDC were generated from bone marrow precursors, as previously described (36). Briefly, bone marrow cells from C57BL/6 or TAP–/– mice were harvested and plated at 2 x 105 cells/ml in CM with 1% of a GM-CSF-containing supernatant. After 3 days of culture at 37°C, medium was added to the dishes. The nonadherent and semiadherent cells were recovered at day 7 or 8 by flushing the plates with PBS EDTA (5 mM). These cells usually contained 60–70% of CD11c+CD11b+ cells that were CD40low and CD86low.

Ag presentation assays

The stimulation of CRMC3 or B3Z T cell hybridoma (105 cells/well) by BMDC (105 cells/well) was monitored by IL-2 release in the supernatants of 18-h cell cultures in 96-well plates. BMDC were pulsed for 4–5 h with proteins or peptides at various concentrations and washed three times before adding 105 T cell hybridoma in 0.2 ml of CM. After 18 h, culture supernatants were frozen for at least 2 h at –80°C. Then 104 cells/well of the IL-2-dependent CTL-L cell line were cultured with 100 µl of these supernatants. After 48 h, [3H]thymidine (50 µCi/ml; Valeant Pharmaceuticals, Orsay, France) was added to the wells. The cells were harvested 6 h later with an automated cell harvester (Molecular Devices, Lier, Norway). Incorporated thymidine was detected by scintillation counting. All assays were done in duplicate.

Inhibitors and Abs

Cycloheximide (CHX, used at 5 µg/ml), brefeldin A (BFA, 5 µg/ml), cytochalasin B (CCB, 5 µg/ml), leupeptin (50 µg/ml), pepstatin (40 µg/ml), chloroquine (CCQ, 50 and 150 µM), N-acetyl-L-leucinal-L-norleucinal (llnl, 12 µg/ml), N-acetyl-L-leucinal-L-methioninal (llml, 12 µg/ml), and chlorpromazine (5 µg/ml) were from Sigma-Aldrich. Lactacystin (BIOMOL, Research Labs, Plymouth Meeting, PA) was used at 1 µM final. The purified mAb specific for murine CD11b (M1/70, rat IgG2b,{kappa}) and the corresponding isotype control were purchased from BD Pharmingen (Le Pont de Claix, France) and used at 10 µg/ml.

Inhibition studies

BMDC were incubated with the drugs or Ab for 1 h in 0.1 ml of CM at 37°C. Then Ag were added in 0.1 ml of CM at optimal concentrations (7.5 nM CyaA-MalE-OVA, OVA257–264, and MalE100–114 peptides, 750 nM MalE protein), in the continuous presence of the inhibitors. In the assays using Ab, the cells were washed three times after 5 h of incubation with both Ag and Ab, and 105 T cell hybridomas were added. In the assays using drugs, the BMDC were washed after the 5-h incubation and fixed using 0.05% glutaraldehyde for 2 min at 37°C (Sigma-Aldrich) and 0.2 M lysine (Sigma-Aldrich). After washing, the T cell hybridomas were added to the wells in 0.2 ml of CM.

For K+ depletion following hypotonic shock, BMDC were incubated for 30 min in serum-free synthetic OptiMEM medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 5 x 10–5 M 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. BMDC were then incubated for 5 min in hypotonic medium (OptiMEM medium and ultrapure H2O, 50/50) and finally for 30 min in K+-free (140 mM NaCl, 20 mM HEPES-NaOH, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml glucose, and 0.5% BSA) or K+-containing buffer (10 mM KCl, 130 mM NaCl, 20 mM HEPES-NaOH, 1 mM CaCl2, 1 mM MgCl2, and 0.5% BSA). Ag were then added to the BMDC at the concentrations indicated in the previous paragraph. BMDC were washed 45 min later, and CM was added for 4 h to allow Ag processing. BMDC were washed, counted, distributed into 96-well plates (105 cells/well), and fixed, as described previously. T cell hybridomas were added to the wells for 18 h.

For all the inhibition studies, the results are expressed as the percentage ± SD of T cell activation in the presence of the inhibitors as compared with T cell activation in the absence of inhibitors. For each Ag, the level of CTL-L proliferation in the absence of inhibitor was considered as 100% of T cell activation. All of the experiments were performed in duplicate.

Mouse immunization

C57BL/6, MHC II–/–, or C57BL/6 x 129sv F1 mice were i.v. injected with 50 µg of CyaA-OVA, CyaA-MalE, CyaA-MalE-OVA, or CyaA E5 diluted in 0.1 ml of PBS.

In vitro cytotoxicity assays

Splenocytes from immunized mice were recovered 7 days after CyaA injection and in vitro restimulated for 5 days with OVA257–264 peptide (1 µg/ml) in the presence of syngeneic irradiated naive spleen cells. The cytotoxic activity was determined in a 5-h in vitro 51Cr release assay, as previously described (16). Briefly, EL-4 (H-2b) tumor cells, loaded or not with 50 µM OVA257–264 peptide, were used as target cells for H-2b effector cells. Various E:T ratios were used, and all assays were performed in duplicate. 51Cr release was counted using a MicroBeta Trilux liquid scintillation counter (Wallac, Turku, Finland). Percentage of specific lysis was calculated as 100 x (experimental release – spontaneous release)/(maximal release – spontaneous release). Maximum release was obtained by adding 10% Triton X-405 to target cells, and spontaneous release was determined with target cells incubated in CM.

Cytokine ELISA

Splenocytes from immunized mice were restimulated in vitro in the presence or absence of 10 µg/ml MalE100–114 or 1 µg/ml OVA257–264 peptide. The culture supernatants were harvested 72 h later, and their IL-4, IL-5, and IFN-{gamma} contents were measured by a standard sandwich ELISA. Maxisorp plates (Nunc, Roskilde, Denmark) were coated with unconjugated anti-IL-4, anti-IL-5, or anti-IFN-{gamma} capture mAb (BVD4-1D11, TRFK5, R4-6A2 clones, respectively; BD Pharmingen), and detection was performed using corresponding biotinylated mAb (BVD6-24G2, TRFK4, XMG1.2 clones; BD Pharmingen). The plates were developed using streptavidin-HRP (BD Pharmingen) and o-phenylenediamine (Sigma-Aldrich) as substrate. All dosages were performed in duplicate. The assays were standardized with recombinant murine cytokines (BD Pharmingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CyaA-MalE is 100 times more efficient than the MalE protein in the delivery of the MalE100–114 CD4+ T cell epitope into the MHC II presentation pathway

We have previously shown that CyaA-MalE delivers the MalE100–114 epitope inserted in its AC domain into the MHC II presentation pathway (31). In this study, we next evaluated the efficiency of this delivery as compared with the entire MalE protein. Using BMDC, which are CD11b positive (data not shown), the presentation of CyaA carrying the MalE100–114 CD4+ T cell epitope inserted at position 108 was compared with MalE protein. APC were incubated with serial dilutions of each protein, and I-Ab-MalE100–114 complex apparition at their surface was monitored with CRMC3, a CD4+ T cell hybridoma specific for this MHC-peptide complex (30). As expected (Fig. 1A), BMDC incubated with CyaA-MalE efficiently stimulated IL-2 secretion by CRMC3. Moreover, a 100 times higher concentration of MalE protein was required to reach the same level of T cell hybridoma stimulation as with CyaA-MalE. As previously shown with splenocytes (31), a 10-fold higher concentration of the free MalE100–114 peptide was necessary to reach the same efficiency as CyaA-MalE at stimulating CRMC3. To exclude that this potentiation was due to a nonspecific stimulatory effect of the CyaA or of some component in the CyaA preparation, BMDC were incubated with various concentrations of MalE100–114 peptide or MalE protein in the presence of control CyaA E5 or of the unrelated construct CyaA-OVA, respectively. As shown in Fig. 1B, no potentiation of MalE100–114 peptide or MalE protein presentation was observed in the presence of CyaA E5 or CyaA-OVA. Thus, the enhancement of MHC II-restricted presentation of the MalE100–114 CD4+ T cell epitope delivered by CyaA was not due to BMDC activation by CyaA itself or a contaminant within the CyaA preparation.



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  		FIGURE 1. Delivery of CyaA-MalE-OVA to both MHC I and II pathways. A and C, BMDC from C57BL/6 mice were incubated for 5 h with various concentrations of CyaA-MalE, CyaA-MalE-OVA, CyaA-OVA, CyaA E5, MalE protein, MalE100–114, or OVA257–264 peptide. After incubation, BMDC were washed, and CRMC3 (A) or B3Z T cell hybridomas (C) were added to the wells. B, BMDC were simultaneously incubated with 7.5 nM CyaA E5 or CyaA-OVA and with various concentrations of MalE100–114 peptide or protein. Five hours later, the cells were washed and 105 CRMC3 T cell hybridomas were added to the wells. A–C, The amounts of IL-2 secreted by CRMC3 or B3Z T cell hybridoma during the 18-h culture were monitored using the IL-2-dependent CTL-L cell line. The results are expressed in cpm ± SD and are representative of at least two experiments.

 
CyaA-MalE-OVA simultaneously delivers CD4+ and CD8+ T cell epitopes for MHC I and II presentation

We have previously reported that CyaA carrying three different CD8+ T cell epitopes could simultaneously induce specific and protective CTL responses against all these three epitopes in vivo (18). In this study, we wanted to determine whether CyaA could deliver both a CD4+ and a CD8+ T cell epitope to BMDC for Ag presentation to the respective specific T cell hybridoma. Therefore, CyaA-MalE-OVA, bearing both the CD4+ MalE100–114 and the CD8+ OVA257–264 T cell epitopes, was compared with CyaA-MalE and CyaA-OVA in a presentation assay. To make this comparison possible, the MalE100–114 CD4+ T cell epitope was inserted between aa 108 and 109 of CyaA-MalE and CyaA-MalE-OVA. The OVA257–264 CD8+ T cell epitope was inserted between aa 336 and 337 in CyaA-OVA and CyaA-MalE-OVA. To detect the presence of Kb-OVA257–264 complexes on BMDC, we used B3Z, a CD8+ T cell hybridoma specific for the OVA257–264 peptide (35). As shown in Fig. 1, A and C, BMDC incubated with CyaA-MalE-OVA stimulated both CRMC3 and B3Z T cell hybridoma. Moreover, CyaA-MalE-OVA was as efficient as CyaA-MalE and CyaA-OVA in the delivery of the MalE100–114 and OVA257–264 epitopes into their respective presentation pathways. Thus, CyaA simultaneously delivers CD4+ and CD8+ T cell epitopes inserted in its AC domain to MHC I and II molecules, and the efficiency of delivery of one epitope is not affected by the insertion of a second epitope at another site within the same AC domain. Indeed, the potentiation of MHC II presentation was still observed with the CyaA bearing both OVA257–267 and MalE100–114 epitopes.

The interaction of CyaA with CD11b on BMDC is required for the enhanced delivery of the reporter CD4+ T cell epitope

The potentiation of MHC II-restricted presentation on CyaA delivery could be explained by the specific interaction of this protein with its CD11b receptor (12, 13), which is expressed on BMDC. To test this hypothesis, BMDC were incubated with 10 µg/ml of either anti-CD11b mAb or isotype control mAb and with Ag. As shown in Fig. 2A, incubation of the APC with anti-CD11b mAb totally and specifically abrogated the presentation of MalE100–114 epitope to CRMC3 following CyaA-MalE-OVA delivery. As expected, the incubation of BMDC with the mAb did not affect the presentation of the MalE protein to the hybridoma. We further confirmed that BMDC incubation with anti-CD11b prevents the generation of Kb-OVA257–264 complexes from CyaA-OVA-MalE (Fig. 2B) without affecting the free OVA257–264 peptide presentation to B3Z. This shows that the high efficiency of CyaA-mediated delivery of epitopes into both the MHC I and II pathways depends on the specific interaction of CyaA with CD11b.



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  		FIGURE 2. Anti-CD11b mAb blocks the delivery of CyaA-MalE-OVA to MHC I and II molecules. BMDC were incubated with 10 µg/ml anti-CD11b or isotype control mAb for 1 h. Ag were then added (see Materials and Methods) to the BMDC in the constant presence of the mAb. DC were washed after 4–5 h of incubation with the Ag, and 105 CRMC3 (A) or B3Z (B) T cell hybridomas were added for 18 h. The supernatants were tested for IL-2 content with the CTL-L cell line. The results are expressed in cpm ± SD and are representative of four experiments.

 
MalE100–114 peptide delivery into the MHC II presentation pathway by CyaA-MalE-OVA does neither require proteasome nor TAP transporter activity

Previous studies have demonstrated that CyaA interaction with CD11b can result in the direct translocation of the AC domain into target cell cytosol. The subsequent processing of this domain to generate peptides for MHC I-restricted presentation requires proteasome and TAP transporters (37). It has been reported that some endogenous Ag are processed in the cytosol for MHC II presentation by an alternative pathway that requires the proteasome and calpain. The peptides released in the cytosol are then transported into endocytic compartments by a poorly understood mechanism (38). Due to the translocation of AC domain into the cytosol, such mechanisms could be responsible for the delivery of the epitopes into the MHC II pathway. Therefore, we tested whether the proteasome is required for CyaA-MalE-OVA delivery to MHC II pathway. BMDC were incubated for 1 h with lactacystin, a 20 S proteasome inhibitor (39, 40), and the Ag were then added. As shown in Fig. 3A, the inhibition of proteasome activity did not abrogate I-Ab-MalE100–114 complex formation and presentation to CRMC3 when CyaA-MalE-OVA, MalE100–114 peptide, or MalE protein were used as Ag. By contrast, OVA257–264 epitope presentation after CyaA-MalE-OVA delivery was totally abrogated by lactacystin (Fig. 3B). We then compared the effect of N-acetyl-L-leucinal-L-norleucinal (llnl, a cathepsin and proteasome inhibitor) and N-acetyl-L-leucinal-L-methioninal (llml, a cathepsin inhibitor) (41) on MalE100–114 peptide presentation to CRMC3 by BMDC. As shown in Fig. 3A, both inhibitors prevented MalE100–114 peptide presentation upon CyaA-MalE-OVA delivery, demonstrating the requirement for cathepsin L or B in this processing pathway. As a control, llnl, but not llml, prevented OVA257–264 peptide presentation to B3Z following CyaA-MalE-OVA delivery. Thus, upon entry into BMDC, the AC domain can be processed to generate peptides for MHC II-restricted presentation by a mechanism that does not require proteasome activity, but requires the activity of cathepsin L or B, two cysteine proteases of the endocytic pathway.



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  		FIGURE 3. CyaA-MalE-OVA delivery to the MHC II pathway does not require proteasome activity nor TAP transporters. A and B, BMDC were incubated for 1 h with 1 µM lactacystin or 12 µg/ml llnl or llml. The Ag were then added at optimal concentrations and 5 h later BMDC were washed and fixed. A total of 105 CRMC3 (A) or B3Z (B) T cell hybridomas was added to the wells for 18 h. The IL-2 content in the culture supernatants was determined with CTL-L cells. Results are expressed in percentage of T cell activation and are representative of two experiments. C and D, The requirement for TAP transporters was determined with TAP–/– BMDC. The BMDC were incubated with various concentrations of Ag and cultured with CRMC3 (C) or B3Z (D) T cell hybridoma. IL-2 production by the hybridomas was determined as previously. The results are expressed in cpm ± SD and are representative of two experiments.

 
To test the TAP requirement for CyaA presentation by MHC II molecules, TAP–/– BMDC were used in a presentation assay to CRMC3. As shown in Fig. 3C, CyaA-MalE-OVA efficiently delivered the MalE100–114 epitope into the MHC II presentation pathway of both wild-type and TAP–/– BMDC. As expected, free MalE100–114 peptide and MalE protein presentation was not dependent on TAP transporters. As previously shown for CyaA-OVA (37), CyaA-MalE-OVA delivery into the MHC I presentation pathway was also dependent on TAP transporters (Fig. 3D). These results demonstrate that CyaA can efficiently deliver its AC domain into two distinct pathways of processing, for the generation of MHC I- and II-restricted epitopes, respectively.

CyaA-MalE-OVA processing for MHC II presentation requires endosomal proteases and vacuolar acidification

After internalization of exogenous Ag, peptide ligands for MHC II presentation are generated in endosomes and lysosomes by a set of proteases that are sequentially activated (2). As cathepsin activity was required to generate MalE100–114 epitope presentation after CyaA-MalE-OVA delivery (Fig. 3A), we tested whether other endocytic proteases are required for I-Ab-MalE100–114 complex formation. Leupeptin (42), an inhibitor for serine and cysteine proteases, and pepstatin (42, 43), an inhibitor for aspartate proteases, inhibited MalE100–114 epitope presentation to CRMC3 when CyaA-MalE-OVA and MalE protein were used as Ag, but did not affect the free peptide presentation (Fig. 4A). It should be mentioned that serine proteases may indeed be required for processing of the N-terminal end of the MalE100–114 epitope. Thus, several endocytic proteases are involved in the AC domain degradation for MHC II peptide generation.



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  		FIGURE 4. CyaA-MalE-OVA delivery into the MHC II pathway requires endocytic protease activity and vacuolar acidification. BMDC were incubated with leupeptin (50 µg/ml), pepstatin (40 µg/ml), or CCQ (50 and 150 µM) for 1 h, and the Ag were added to the wells at optimal concentrations. After washing and fixation of the BMDC, 105 CRMC3 (A) or B3Z (B) T cell hybridomas were added. The culture supernatants were harvested 18 h later, and their IL-2 content was determined with CTL-L. The results are representative of two to five experiments.

 
Vacuolar acidification was previously shown to control the sequential activation of endocytic proteases. Therefore, we used CCQ, an inhibitor of endocytic vesicle acidification, to confirm that MalE100–114 epitope presentation following CyaA delivery occurs after endocytic processing. As shown in Fig. 4A, CCQ strongly diminished the presentation of the MalE100–114 epitope following both CyaA-MalE-OVA and MalE protein delivery, whereas free peptide presentation remained unaffected. Thus, the processing of the AC domain by BMDC yielding MHC II-restricted peptides is dependent on vacuolar acidification and endocytic proteases. As expected, presentation of the OVA257–264 epitope was, however, not dependent on vacuolar acidification when CyaA-MalE-OVA was used as Ag (Fig. 4B) (37). These results suggest that after binding to CD11b, the CyaA AC domain can either be translocated into BMDC cytosol to be further processed by proteasome or can be captured in vesicles to enter the endocytic route of processing. These results, however, do not exclude the possibility that some of the endocytosed CyaA molecules may still be capable of translocating their AC domain to the cytosol across the endosomal membrane.

MalE100–114 epitope delivery by CyaA-MalE-OVA is sensitive to the inhibition of protein synthesis by CHX and Golgi disruption by BFA

Generation of MHC II-peptide complexes along the conventional endocytic pathway requires peptide loading to newly synthesized MHC II molecules (2). To determine whether I-Ab-MalE100–114 complex generation after CyaA-MalE-OVA delivery requires nascent MHC II molecules, we used CHX, an inhibitor of protein synthesis. As shown in Fig. 5A, BMDC that have been preincubated with CHX before addition of CyaA-MalE-OVA or MalE protein did not stimulate IL-2 secretion by CRMC3. Moreover, OVA257–264 peptide presentation to B3Z following CyaA-MalE-OVA delivery was also totally abrogated by CHX (Fig. 5B). As expected, CHX did not inhibit the presentation of the free peptides to these T cell hybridomas. Thus, newly synthesized proteins are necessary for both MHC I and MHC II-restricted presentation of the CyaA AC domain. To determine whether MHC II molecules that present the MalE100–114 peptide reach early and late endosomes toward Golgi, we used BFA, an inhibitor of Golgi transport (44, 45). The presentation of the MalE100–114 epitope after its delivery to BMDC by CyaA-MalE-OVA or MalE protein was totally abrogated by BFA (Fig. 5A), while free peptide presentation was not affected. Thus, de novo synthesis of MHC II molecules and trafficking through Golgi are necessary for BMDC to present the MalE100–114 epitope delivered by CyaA-MalE-OVA.



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  		FIGURE 5. CyaA-MalE-OVA delivery into MHC I and II pathways requires protein neosynthesis and Golgi transport. BMDC were incubated for 1 h with CHX (5 µg/ml) or BFA (5 µg/ml), and Ag were added at optimal concentrations. After 5 h, the cells were washed and fixed. CRMC3 (A) or B3Z (B) T cell hybridomas were added to the wells, and the IL-2 contents in 18-h culture supernatants were monitored with the CTL-L cell line. The results are representative of four experiments.

 
MalE100–114 epitope delivery by CyaA-MalE-OVA does not depend on actin filament polymerization, but requires clathrin-coated pits

The internalization of CyaA and the subsequent MHC I-restricted presentation of the OVA257–264 epitope inserted in its AC domain have been shown to be independent on phagocytosis (37). However, it cannot be excluded that some CyaA molecules are captured and processed as classical exogenous Ag to give rise to MHC II presentation. We thus tested whether actin-dependent capture was implicated in the MalE100–114 epitope delivery to the MHC II-restricted presentation pathway. We used CCB, a drug that prevents actin filament polymerization and impairs macropinocytosis, phagocytosis, and also caveolae-mediated endocytosis (46). As expected, while the free MalE100–114 peptide was still presented to CRMC3, presentation of the MalE protein was totally abrogated by CCB. In contrast, upon delivery by the CyaA-MalE-OVA protein, neither the presentation of the MalE100–114 epitope, nor that of the OVA257–264 epitope to their respective specific T cell hybridomas was inhibited by treatment with CCB, as shown in Fig. 6. It can, hence, be concluded that the AC domain-mediated delivery of epitopes to MHC I and II molecules does not involve phagocytosis, macropinocytosis, or caveolae-mediated endocytosis of CyaA by BMDC.



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  		FIGURE 6. MHC II epitope delivery by CyaA-MalE-OVA does not require phagocytosis, but is dependent on clathrin-mediated endocytosis. BMDC were incubated with CCB (5 µg/ml) or chlorpromazine (5 µg/ml) for 1 h at 37°C and the Ag were added at the optimal concentrations. After 5 h of incubation, BMDC were washed three times and fixed. For K+ depletion, cells were incubated in serum-free medium, submitted to a hypotonic shock, and then incubated with the optimal concentrations of Ag for 45 min in the absence of K+ ions. Cells were washed, incubated for 4 h in CM, and fixed. CRMC3 (A) or B3Z (B) T cell hybridomas were added at 105 cells/well for 18 h. The supernatants were tested for IL-2 content with the CTL-L cell line. The results are representative of two to four experiments.

 
As CyaA interacts with CD11b on APC cell surface, it could be endocytosed by a clathrin-dependent process. We used K+ depletion following hypotonic shock (47, 48, 49) and inhibition by chlorpromazine to test whether clathrin-coated pits were required for MHC I and II presentation of CyaA-MalE-OVA. K+ depletion following hypotonic shock was performed by BMDC exposure to hypotonic medium, followed by incubation in the absence of extracellular potassium. This treatment results in dissociation of clathrin coats from the plasma membrane and leads to nonproductive assembly of clathrin cages in the cytoplasm. Internalization of membrane proteins that interact with AP2 clathrin adapter complex through cytoplasmic amino acid sequences is therefore impaired. As shown in Fig. 6A, MalE100–114 epitope presentation to CRMC3 T cell hybridoma after CyaA-MalE-OVA delivery was totally abrogated by K+ depletion, whereas presentation of the free MalE peptide was not inhibited. Furthermore, presentation of the OVA257–264 epitope upon delivery by CyaA-MalE-OVA was also not abrogated by this treatment (Fig. 6B). To confirm this result, we used chlorpromazine, a cationic amphiphilic drug that has been widely used to analyze virus entry into target cells by clathrin-mediated endocytosis (50, 51). MalE100–114 epitope, but not OVA257–264 epitope, presentation was inhibited by chlorpromazine after BMDC incubation with CyaA-MalE-OVA (Fig. 6). Importantly, MalE protein presentation was not impaired by chlorpromazine treatment, showing that MHC II-restricted presentation by these cells was still possible. Thus, endocytosis of CyaA-MalE-OVA by a clathrin-mediated mechanism was required for MHC II-, but not for MHC I-restricted presentation.

CyaA-MalE-OVA simultaneously induces OVA-specific CD8+ T cell responses and MalE-specific CD4+ T cell responses in vivo

We have previously demonstrated the efficacy of CyaA in inducing CTL responses against different CD8+ T cell epitopes (18) and in stimulating proliferative CD4+ T cell responses against a MalE epitope (31). Having shown in this study that CyaA is a potent vehicle to simultaneously deliver both the CD4+ and CD8+ T cell epitopes to BMDC for presentation in vitro, we tested the efficiency of CyaA-MalE-OVA in the simultaneous delivery of these epitopes in vivo. C57BL/6 mice were therefore immunized with 50 µg of CyaA-MalE, CyaA-MalE-OVA, CyaA-OVA, or CyaA E5 by i.v. route, respectively, without adjuvant, and the T cell responses were assessed 7 days after injection.

As readout for CD8+ T cell responses, the cytotoxic activity of splenocytes from immunized mice was tested using target cells loaded with the OVA257–264 peptide. As shown in Fig. 7A, both CyaA-MalE-OVA and CyaA-OVA induced specific CTL responses against the OVA257–264 epitope. As expected, no response was detected when mice received CyaA-MalE or CyaA E5. Following immunization by the CyaA-OVA or CyaA-MalE-OVA, a Th1-like polarized OVA-specific T cell response was observed upon in vitro restimulation with the OVA257–264 peptide, which was characterized by a strong IFN-{gamma} production, but no IL-5, IL-4, or IL-10 secretion (Fig. 7B, and data not shown). Thus, in vivo, CyaA-MalE-OVA and CyaA-OVA were equally potent in induction of a Th1-polarized CD8+ T cell response. Moreover, both CyaA-OVA and CyaA-MalE-OVA induced strong CTL responses in MHC II–/– mice, showing that CyaA presentation to CD8+ T cells results in their activation even in the absence of CD4+ Th cells (Fig. 7D). Control 129sv x C57BL/6 mice developed similar CTL responses as C57BL/6 mice (data not shown).



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  		FIGURE 7. Immunization by CyaA-MalE-OVA induces both CD4+ and CD8+ T cell responses. A–C, Splenocytes of C57BL/6 mice i.v. injected with 50 µg of CyaA-MalE, CyaA-MalE-OVA, CyaA-OVA, or CyaA E5 were harvested 1 wk after immunization. A, Splenocytes were stimulated for 5 days in the presence of 1 µg/ml OVA257–264 peptide and tested for CTL activity on 51Cr-labeled EL-4 target cells incubated or not with the same peptide. Spontaneous cell 51Cr release was obtained with EL-4 incubated in medium alone. Each curve represents the CTL response of a single mouse representative of four (CyaA E5) to eight mice (CyaA-MalE, CyaA-OVA, CyaA-MalE-OVA) tested in four different experiments. B and C, Splenocytes were stimulated for 72 h in the presence or absence of 1 µg/ml OVA257–264 peptide (B) or 10 µg/ml MalE100–114 peptide (C). The culture supernatants were tested for IL-5 and IFN-{gamma} content in an ELISA. Results represent the difference between the cytokine concentration in the presence and absence of the peptide and are representative of four experiments. D, MHC II–/– mice were i.v. immunized with 50 µg of CyaA E5, CyaA-MalE, CyaA-OVA, or CyaA-MalE-OVA and sacrificed 7 days later. Splenocytes were in vitro stimulated with 1 µg/ml OVA257–264 peptide for 5 days, and CTL activity was assayed, as previously described. Each curve represents the CTL response of a single mouse representative of two to six mice tested in two independent experiments.

 
We further analyzed the CD4+ T cell responses induced by CyaA-MalE-OVA as compared with CyaA-MalE. As shown in Fig. 7C, both CyaA-MalE-OVA and CyaA-MalE induced a strong and specific IFN-{gamma} production by immune splenocytes. IL-5 secretion was also detected in three experiments of four, although at very low levels (Fig. 7C), showing that the CD4+ T cell responses induced by CyaA were mainly Th1 polarized. No IL-10 or IL-4 was, indeed, detectable in the supernatants of splenocyte cultures. These results demonstrate the capacity of CyaA to deliver simultaneously both CD4+ and CD8+ T cell epitopes for in vivo T cell priming. The efficiency of such simultaneous delivery was similar to the delivery of the single epitopes, and both CD4+ and CD8+ T cell responses remained Th1 polarized.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate the capacity of genetically detoxified CyaA to simultaneously deliver CD4+ and CD8+ T cell epitopes into CD11b-expressing DC. We show that the insertion of both a CD4+ and a CD8+ T cell epitope at two different permissive sites within the cell-invasive AC domain does not affect the capacity of such CyaA construct to deliver each of these epitopes into its respective cytosolic, or endosomal Ag presentation pathway. The present results demonstrate that besides the previously characterized capacity of CyaA to deliver the CD8+ T cell epitopes into the MHC class I-restricted pathway, this carrier is also a potent vehicle for efficient delivery of CD4+ T cell epitopes into the endosomal pathway, yielding MHC II-restricted presentation both in vitro and in vivo.

The delivery of epitopes by CyaA into both the MHC I- and II-restricted Ag presentation pathways required the interaction of CyaA with CD11b (12, 15). Detailed characterization of the mechanisms underlying the entry of CyaA into target cells and delivery of the cargo epitopes for presentation allowed corroboration of the previous observations that CyaA can translocate its AC domain into target cell cytosol directly across the plasma membrane without the need for endocytosis (11, 21, 52). Indeed, our results are in agreement with several studies that have demonstrated that CyaA in vitro intoxicates cells independently of any vesicular trafficking, and that this intoxication is very fast (52). However, our results show that unlike MHC I-restricted presentation, MHC II-restricted presentation of CyaA depends on its internalization through a clathrin-mediated endocytosis. This endocytosis of CyaA after its interaction with CD11b is likely to be mediated by the CD18 subunit of the {alpha}M{beta}2 integrin, which bears two putative NPFX internalization sites in its cytoplasmic tail (53). Therefore, our results support the conclusion that after CyaA binding to CD11b, its AC domain can either be directly translocated from cell membrane into the cytosol to reach the cytosolic processing route or be taken up into vesicles for endocytic degradation.

Detailed analysis of the mechanisms implicated in the presentation of the CyaA-delivered MalE epitope shows that the processing of the AC domain for MHC II-restricted presentation required involvement of endocytic proteases activated by vesicle acidification and de novo synthesis of MHC II molecules. Thus, the AC underwent processing along the conventional endocytic route for MHC II-restricted presentation. These results demonstrate that the CyaA AC domain reaches two distinct processing compartments after entering the cell by two distinct routes.

It is worth noting that upon CD11b-targeted delivery of the epitope by CyaA, the in vitro efficiency of the resulting MHC II-restricted presentation of the MalE100–114 CD4+ T cell epitope was enhanced by ~2–3 orders of magnitude, as compared with the presentation of equivalent amounts of the MalE protein. Similar enhancement of both MHC I- and II-restricted presentation has also been previously reported upon targeting of Ag to another DC receptor, CD205, using specific mAb. However, while presentation of peptides associated to CD205 mAb is efficient, this delivery procedure rather leads to induction of tolerance than to immunity, except when an additional DC maturation signal is provided (54, 55, 56). By contrast, a single injection of 50 µg of CyaA by i.v. route, without adjuvant, could induce both Th1-biased CD4+ T cell response and CTLs in vivo. Moreover, CTL responses were still induced following injection of CyaA in MHC II–/– mice, excluding the possibility that indirect activation of DC by CD4+ Th cells is responsible for the high immunogenicity of CyaA. This suggests that in addition to allowing the delivery of epitopes into DC, the interaction of CyaA with CD11b may promote maturation of DC by some as yet uncharacterized signaling mechanism. We have previously shown that among the CD11b+ cells, the CD8{alpha}– DC subset accounts for the in vivo presentation of epitopes delivered by CyaA (15). This DC subset has been suggested to promote Th2- rather than Th1-type T cell priming (57), while it is shown in this study that the CD4+ T cell responses induced in vivo by CyaA are strongly polarized toward Th1 profile. This suggests that CyaA may be harnessing DC toward a Th1-polarizing state of activation, and experiments are underway to elucidate the mechanisms by which CyaA promotes Th1 polarization of induced responses.

The simultaneous induction of robust Th1 CD4+ and CTL T cell responses is an important aim of vaccination. As targeting of CD11b+ DC by CyaA leads to the enhancement of MHC II-restricted presentation of delivered epitopes and to the induction of Th1-polarized T cell responses in parallel to an efficient MHC I-restricted presentation of epitopes delivered simultaneously, the CyaA appears to be a very attractive Ag carrier for future vaccine design.


   Acknowledgments
 
We thank Daniel Ladant and J. M. Clement for the gift of CyaA E5 and MalE proteins.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This project was supported by grants from the European Community (FP6 503582, Theravac), Association pour la Recherche sur le Cancer, and Agence Nationale de Recherches sur le SIDA. G.S. was supported by a PhD fellowship from the French government (Ministére de la Recherche et de l’Espace). J.L. was supported by Grant S5020311 from the Czech Academy of Sciences. Back

2 Address correspondence and reprint requests to Dr. Claude Leclerc, Biologie des Régulations Immunitaires, Institut National de la Santé et de la Recherche Médicale E 352, Institut Pasteur, 25 rue du Docteur Roux 75724 Paris cedex 15, France. E-mail address: cleclerc{at}pasteur.fr Back

3 Abbreviations used in this paper: CyaA, B. pertussis adenylate cyclase; AC, adenyl cyclase; BFA, brefeldin A; DC, dendritic cell; BMDC, bone marrow-derived DC; CCB, cytochalasin B; CCQ, chloroquine; CHX, cycloheximide; CM, complete medium; llml, N-acetyl-L-leucinal-L-methioninal; llnl, N-acetyl-L-leucinal-L-norleucinal. Back

Received for publication June 10, 2004. Accepted for publication September 15, 2004.


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G. Y. C. Cheung, D. Xing, S. Prior, M. J. Corbel, R. Parton, and J. G. Coote
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M. Skoberne, S. Somersan, W. Almodovar, T. Truong, K. Petrova, P. M. Henson, and N. Bhardwaj
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